Cleavage specificity of a proteolytic antibody light chain and effects of the heavy chain variable domain. 1997

M Sun, and Q S Gao, and L Kirnarskiy, and A Rees, and S Paul
Department of Anesthesiology and Eppley Cancer Research Institute, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198-6830, USA.

The recombinant light chain (L chain) of an antibody raised by immunization with vasoactive intestinal polypeptide (VIP) cleaved this peptide on the C-terminal side of basic residues. The major sites of cleavage in VIP were two adjacent peptide bonds, Lys20-Lys21 and Lys21-Tyr22. Lower levels of cleavage were evident at Arg14-Lys15 and Lys15-Gln16. Hydrolysis of radiolabeled VIP by the L chain was inhibited by two serine protease inhibitors, diisopropylfluorophosphate and aprotinin, but not by soybean or lima bean trypsin inhibitors or inhibitors of other classes of proteases. To probe the role of the VH domain, single chain Fv constructs composed of the VL domain of the anti-VIP L chain linked via a 14-residue peptide to its natural VH domain partner or an irrelevant anti-lysozyme VH domain (hybrid Fv) were prepared. The anti-VIP Fv hydrolyzed VIP with Ks 21.4-fold lower than the L chain and 250-fold lower than the hybrid Fv, suggesting increased affinity for the substrate ground state due to the anti-VIP VH domain. The kinetic efficiency (kcat/Ks) of the anti-VIP Fv was 6.6-fold greater compared to the L chain and 29.4-fold greater compared to the hybrid Fv. Peptide-MCA substrates unrelated in sequence to VIP were hydrolyzed by the anti-VIP Fv and L chain at equivalent rates. These observations lead to a model of catalysis by the anti-VIP Fv in which the essential catalytic residues are located in the VL domain and additional residues from the VH domain are involved in high affinity binding of the substrate.

UI MeSH Term Description Entries
D007128 Immunoglobulin Fragments Partial immunoglobulin molecules resulting from selective cleavage by proteolytic enzymes or generated through PROTEIN ENGINEERING techniques. Antibody Fragment,Antibody Fragments,Ig Fragment,Ig Fragments,Immunoglobulin Fragment,Fragment, Antibody,Fragment, Ig,Fragment, Immunoglobulin,Fragments, Antibody,Fragments, Ig,Fragments, Immunoglobulin
D007135 Immunoglobulin Variable Region That region of the immunoglobulin molecule that varies in its amino acid sequence and composition, and comprises the binding site for a specific antigen. It is located at the N-terminus of the Fab fragment of the immunoglobulin. It includes hypervariable regions (COMPLEMENTARITY DETERMINING REGIONS) and framework regions. Variable Region, Ig,Variable Region, Immunoglobulin,Framework Region, Immunoglobulin,Fv Antibody Fragments,Fv Fragments,Ig Framework Region,Ig Variable Region,Immunoglobulin Framework Region,Immunoglobulin Fv Fragments,Immunoglobulin V,Antibody Fragment, Fv,Antibody Fragments, Fv,Fragment, Fv,Fragment, Fv Antibody,Fragment, Immunoglobulin Fv,Fragments, Fv,Fragments, Fv Antibody,Fragments, Immunoglobulin Fv,Framework Region, Ig,Framework Regions, Ig,Framework Regions, Immunoglobulin,Fv Antibody Fragment,Fv Fragment,Fv Fragment, Immunoglobulin,Fv Fragments, Immunoglobulin,Ig Framework Regions,Ig Variable Regions,Immunoglobulin Framework Regions,Immunoglobulin Fv Fragment,Immunoglobulin Variable Regions,Regions, Immunoglobulin Variable,Variable Regions, Ig,Variable Regions, Immunoglobulin
D007143 Immunoglobulin Heavy Chains The largest of polypeptide chains comprising immunoglobulins. They contain 450 to 600 amino acid residues per chain, and have molecular weights of 51-72 kDa. Immunoglobulins, Heavy-Chain,Heavy-Chain Immunoglobulins,Ig Heavy Chains,Immunoglobulin Heavy Chain,Immunoglobulin Heavy Chain Subgroup VH-I,Immunoglobulin Heavy Chain Subgroup VH-III,Heavy Chain Immunoglobulins,Heavy Chain, Immunoglobulin,Heavy Chains, Ig,Heavy Chains, Immunoglobulin,Immunoglobulin Heavy Chain Subgroup VH I,Immunoglobulin Heavy Chain Subgroup VH III,Immunoglobulins, Heavy Chain
D007147 Immunoglobulin Light Chains Polypeptide chains, consisting of 211 to 217 amino acid residues and having a molecular weight of approximately 22 kDa. There are two major types of light chains, kappa and lambda. Two Ig light chains and two Ig heavy chains (IMMUNOGLOBULIN HEAVY CHAINS) make one immunoglobulin molecule. Ig Light Chains,Immunoglobulins, Light-Chain,Immunoglobulin Light Chain,Immunoglobulin Light-Chain,Light-Chain Immunoglobulins,Chains, Ig Light,Chains, Immunoglobulin Light,Immunoglobulins, Light Chain,Light Chain Immunoglobulins,Light Chain, Immunoglobulin,Light Chains, Ig,Light Chains, Immunoglobulin,Light-Chain, Immunoglobulin
D007531 Isoflurophate A di-isopropyl-fluorophosphate which is an irreversible cholinesterase inhibitor used to investigate the NERVOUS SYSTEM. DFP,Diisopropylfluorophosphate,Fluostigmine,Bis(1-methylethyl) Phosphorofluoridate,Di-isopropylphosphorofluoridate,Diisopropylphosphofluoridate,Dyflos,Floropryl,Fluorostigmine,Di isopropylphosphorofluoridate
D007611 Aprotinin A single-chain polypeptide derived from bovine tissues consisting of 58 amino-acid residues. It is an inhibitor of proteolytic enzymes including CHYMOTRYPSIN; KALLIKREIN; PLASMIN; and TRYPSIN. It is used in the treatment of HEMORRHAGE associated with raised plasma concentrations of plasmin. It is also used to reduce blood loss and transfusion requirements in patients at high risk of major blood loss during and following open heart surgery with EXTRACORPOREAL CIRCULATION. (Reynolds JEF(Ed): Martindale: The Extra Pharmacopoeia (electronic version). Micromedex, Inc, Englewood, CO, 1995) BPTI, Basic Pancreatic Trypsin Inhibitor,Basic Pancreatic Trypsin Inhibitor,Bovine Kunitz Pancreatic Trypsin Inhibitor,Kallikrein-Trypsin Inactivator,Kunitz Pancreatic Trypsin Inhibitor,Trypsin Inhibitor, Basic, Pancreatic,Trypsin Inhibitor, Kunitz, Pancreatic,Antilysin,Bovine Pancreatic Trypsin Inhibitor,Contrical,Contrykal,Dilmintal,Iniprol,Kontrikal,Kontrykal,Pulmin,Traskolan,Trasylol,Zymofren,Inactivator, Kallikrein-Trypsin,Kallikrein Trypsin Inactivator
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010450 Endopeptidases A subclass of PEPTIDE HYDROLASES that catalyze the internal cleavage of PEPTIDES or PROTEINS. Endopeptidase,Peptide Peptidohydrolases
D010455 Peptides Members of the class of compounds composed of AMINO ACIDS joined together by peptide bonds between adjacent amino acids into linear, branched or cyclical structures. OLIGOPEPTIDES are composed of approximately 2-12 amino acids. Polypeptides are composed of approximately 13 or more amino acids. PROTEINS are considered to be larger versions of peptides that can form into complex structures such as ENZYMES and RECEPTORS. Peptide,Polypeptide,Polypeptides
D002384 Catalysis The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction. Catalyses

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